Abstract
A semisynthetic antibiotic rifampicin 3-(4-methyl-piperazinyl-iminomethyl)-rifamycin SV has been noted clinically as a powerful antibacterial drug. But of metabolism of the drug in human body, many problems still remain unknown. A remarkable experimental fact was found that rifampicin was promptly oxidized by treating with human saliva under hydrochloric acid condition. As such oxidation could not be observed in the solution of this acid without saliva, it seemed that a certain enzyme should bring about this oxidation. This is a report about a special oxidation enzyme present in human saliva.
By using rifampicin as a substrate, the salivary enzyme activity could be determined tentatively by a spectrophotometric assay in accordance with the principle that by oxidation of rifampicin the absorption peak is changed from 475 mμ to 540mμ. This enzyme was relatively unstable, being destroyed gradually at 25° However, when stored at 5°, it was possible to keep the enzyme in constant activity for a long time. The enzyme activity was completely inhibited by H2S, cysteine, cyanides, citrates and diethyldithiocarbamate. This is an evidence to show that this enzyme contains a certain heavy metal, perhaps copper which is a conspicuous oxidation catalyst. Methanol, ethanol and propanol were also very strong inhibitors, and it was further observed that pig's pancreatic lipase attacked strongly the activated enzyme under acid condition. These inactivations probably suggest that the enzyme also contains some lipid which is required perhaps for the complete activity.
With rifampicin as a substrate, the complete enzyme activity appeared at a very low pH below 3. In contrast, no activity was observed at a pH over 4. Narrowness of the pH range susceptible to convert the enzyme from inactivity into activity was also remarkable. Such pH dependent activity of this enzyme indicates undoubtedly that the original enzyme in saliva is in an inactive form, and is activated by gastric acid (HCl) when reached the stomach. The salivary enzyme at a neutral pH revealed a complete resistance to heat treatments within 30 minutes. But the activated enzyme in acid saliva was very unstable, so that the enzyme was inactivated rapidly and completely at 40° for 30 minutes, and partially at 37°. In addition, any mechanical treatments seemed to constitute hindrance to the activated enzyme.
When saliva was dialyzed in a cellophane bag against distilled water for one day at 5°, the enzyme activity was recognized in the liquid outside of cellophane bag. The enzyme was eluted in a localized manner in a peptide fraction when saliva had been gel-filtrated with distilled water on a 1.4×15cm Sephadex G-15 column. The enzyme fraction appeared to be of positive ninhydrine reaction and of negative biuret reaction. It may thus be concluded that this enzyme is a very low molecule compound, perhaps consisting of oligopeptide.
A heavy metal, which forms a black metal sulfide by reaction with ammonium sulfide, was detectable apparently in the enzyme fraction while negligible in other fractions. But Cl and urea exerting any questionable influence on the enzyme activity were both negative in the fraction. Paper chromatography of a relatively pure enzyme fraction obtained by gel-filtration revealed only one spot, which was coloured by spray of ninhydrine. However, there have remained further demonstrations to prove that the spot is identical to the enzyme itself.
The specific absorption peak of the enzyme fraction was found around 270mμ. This enzyme was distinguishable from well-known copper enzymes: tyrosinase, ascorbase and polyphenol oxidase, and also peroxidase which is a porphyrin iron compound, in view of comparison of their optimum pH, molecular weight and spectrum one with another.
